Optimising PROTACs against integral membrane proteins

Integral membrane proteins (IMPs) play a critical role in the pathophysiology of a range of diseases. Resistance mechanisms to conventional IMP-targeted therapeutics have driven the need for novel modalities. While PROteolysis TArgeting Chimeras (PROTACs) have shown significant potential for degrading cytosolic proteins, their use in the targeted degradation of IMPs remains challenging due to structural complexity and limited accessibility of IMPs. This thesis explored novel approaches to support the development of IMP-targeted PROTACs. First, potential reasons underlying the complexity of targeting IMPs for PROTAC-mediated degradation were discussed. These included the strong IMP-lipid bilayer interaction and poor understanding of the mechanism of degradation. Next, we addressed current limitations in traditional immunoblotting, which has low throughput and lacks spatial resolution. We proposed an immunofluorescence-based protocol, which provides increased accuracy, a qualitative and quantitative insight into degradation efficacy and a critical distinction between cell membrane-localised and intracellular IMP protein pools. To address the underutilisation of the E3 ligase repertoire, we also developed a ligand-independent E3 ligase recruitment system, integrated with an imaging-based assay, to identify putative E3 ligase candidates. Lastly, we integrated machine learning with proteome-profiling to characterise off target effects of androgen receptor-targeted PROTACs. We demonstrated a link between observed mitochondrial toxicity and the respiratory chain complex I and provided an analytical framework for the proteome-guided rational PROTAC development. Overall, the body of work conducted as part of the PhD thesis provides novel insights into more effective drug development for the targeted degradation of IMPs.